1. Field of the Invention
The present invention relates to a method for analyzing a DNA microarray and a sample using the DNA microarray. More specifically, it relates to technology for improving accuracy and automating image analysis of the DNA microarrays by performing the alignment of a detection area defined by an analyzing tool and image correction relative to images of the DNA microarrays read via a scanner, in the image analysis of the DNA microarrays after hybridization.
2. Description of the Related Art
In recent years, genetic analysis has been implemented using DNA microarrays.
In this specification, DNA microarrays or DNA chips refer to the spotting of DNA in a grid-like pattern upon an array substrate comprising glass or the like.
Upon the DNA microarrays, DNA is spotted as probes capable of specifically reacting to marked DNA samples.
After an unknown DNA sample to be analyzed is run in onto the DNA microarrays and the sample has been given an optically detectable light-emitting or fluorescent mark, if there are parts of the DNA sample that have a complimentary relationship with the DNA upon the grid then those parts of the DNA sample are linked to the DNA on the grid and become double stranded. Those DNA to which the portions of the sample do not link are rinsed away, and the condition of the double-stranded DNA may be observed as images by illuminating the DNA samples to be determined, and reading them with a scanner. In other words, it is possible to analyze the existence of the required genes, whether or not genes appear, or the degree to which the genes appear by analyzing the distribution of marks which emit light on the DNA microarrays. In this manner, a set of DNA is constructed upon the DNA microarray, and then through replacement with the relevant set of DNA, gene mutations and expression levels of genes, etc., may be detected.
FIG. 1 illustrates a sequence of processing in such DNA microarray analysis.
As shown in FIG. 1, in the hybridization step 3, unknown DNA samples that are to be analyzed are given luminescent marks and dropped onto a DNA microarray 1, which includes a spot region 1a upon which the DNA have been mounted. Here, if it is found that the DNA samples have a complementary relationship with the DNA on the grid, they are linked and become double stranded. Next, in the wash step 5, when the hybridized DNA microarrays 1 are washed away with a prescribed cleaning liquid, all DNA which are not linked are washed away. In the scanning step 7, the amount of light emission of each spotted DNA (gene) is measured by directing a laser beam with a prescribed wavelength suitable for exciting the luminescent marks (for example, Cy3, or Cy5) and by scanning the rinsed DNA microarrays within a scanner unit. Images of the scanned DNA microarrays 1 are stored in the DNA microarray image file 11. In the analysis step 9, the DNA microarray image file 11 is read in, and analysis processing of the DNA microarrays is implemented. More specifically, in the analysis step 9, based on the image data that has been read in, the fluorescent intensity of each spot is calculated and various analyses are implemented.
It should be noted that the DNA microarray images, which are read in through the scanner not from the DNA microarray image file 11, may be entered sequentially in the analysis step 9.
The analysis results are recorded as digital data files 13, and may be output in a displayed format through a display unit 15 or output in a printed format through a printing unit 17 as necessary.
FIG. 2 illustrates an example of the DNA microarrays 1 used in DNA microarray analysis. As shown in FIG. 2, the DNA microarray 1 comprises blocks in which individual genes (hereafter, referred to as “spots”) of complementary DNA (cDNA) are respectively arranged upon the grids of the substrate 2 in a matrix with a predetermined number thereof in each row and each column. It should be noted that spots arranged in a spot region 1a on the substrate 2 respectively correspond to DNA, each differing from each other, and having the base sequence thereof already decoded, wherein the arrangement positioning upon the substrate 2 is predetermined.
FIG. 3 illustrates an example of a template, which is applied to the DNA microarray image file 11 during analysis processing. As shown in FIG. 3, the template is divided into a plurality of fields, such as A through F, and there are a plurality of blocks (p×24 in FIG. 3), which are formed by detection areas (corresponding to each spot of the DNA microarray) arranged in a matrix of m-row by n-column (4×4 in FIG. 3), within each field. Address numbers, such as a through p and 1 through 24, are attached to the blocks in both the X and Y directions, and the position of each block can be represented, for example, as “a, 1” in each field.
In the above mentioned analysis step 9, the individual spots in the read DNA microarray scanned image are applied to the detection area of the template provided by the analysis tool. The positioning operation by applying the detection area to the image is referred to as the alignment. In the analysis step 9, the alignment (positioning) process must be performed correctly in order to correctly set the individual detection areas arranged upon the substrate 2 of the DNA microarrays, which is pre-defined for individual spots on the image, so that the analysis tool may calculate the fluorescent intensity of each spot on the DNA microarrays and perform the correct analysis. (In order to analyze the read image correctly, individual detection areas must be applied to the corresponding individual spots during this alignment.) During this alignment (positioning) processing, in the case where a misalignment is detected, the position of an imaging or detection spot must be compensated so as to be set correctly in the detection area of the corresponding template.
However, the conventional method for analyzing the DNA microarrays through the hybridization step 3 has the following problems that need to be solved.
Namely, these problems include the low machined precision of the substrate 2, which is formed from glass and which configures the DNA microarray, and development of chips/cracks and breakage therein; the misalignment that developments when putting the DNA microarrays on the scanner autoloader or when loading them into the scanner; the accidental error built into the mechanical precision of the autoloader, the scanner, and the like; and further, the micro-dust that attaches to the substrate 2 and inevitably leads to misalignment occurring in the image file of the DNA microarrays to be analyzed.
However, external dimensions of this substrate 2 used to configure the DNA microarrays have low accuracy because of the machined precision, chips/cracks, and breakage of the glass. Therefore, in practical use, the outside dimension itself of the substrate 2 cannot be used for aligning the DNA microarrays and template provided by the analysis tool.
Accordingly, multiple DNA microarrays are set onto the autoloader, and the DNA microarrays are continuously scanned by the scanner to automate the accumulation of image data 11. In the following analysis step 9, it is then necessary to manually detect the misalignment caused by misalignment in θ direction (rotational misalignment), misalignment in x direction, and misalignment in y direction, and manually align the detection area for the DNA microarray image file 11, or else determine whether or not the alignment of the detection area performed by the existing analysis apparatus makes an error.
In particular, in cases where misalignment of the image is extreme, the alignment processing cannot be performed in the correct manner using automation.
Accordingly, DNA microarray image file detection area alignment processing and detection area alignment success/failure determination is dependent on human observation and judgment, and the inability to automate this has created a bottleneck in the processing.
In particular, in the case where there is a large number of pages of the images to be analyzed, the alignment processing of these detection areas and determination of whether the alignment has been successful or unsuccessful for the detection area requires a large amount of time and labor. Moreover, as inkjet techniques have been adapted to technology for spot formation upon the DNA microarrays, due to the increased miniaturization of spot diameters as well as increasingly high-pitched intervals between spots, performing proper alignment processing manually or through visual observation has become especially difficult.